We have shown previously the presence of plasma membrane receptors for 3,3',5-triiodo-L-thyronine (T3) in cultured cells. To explore the possibility of using human placenta of Swarm rat chondrosarcoma as the source for the large scale purification of the plasma membrane T3 receptors, the binding of T3 on chondrocytes and the purified plasma membranes of human placenta was characterized. Two classes of speficic T3 binding sites were detected on human placenta and chondrocytes: a high affinity binding site with a Kd of 2.0 nM and 0.5 nm, respectively and a low affinity binding site with a Kd of 18.5 MuM and 0.2 MuM, respectively. By the affinity labeling, peptide mapping and immunoprecipitation, the plasma membrane T3 receptors in human placenta and chondrocytes were shown to be similar to those of cultured cells. These results indicate that either tissue can be used as a source for the purification of T3 receptors. Polyclonal antibodies against the plasma membrane T3 receptors were developed using intact GH3 cells, purified plasma membranes of human placenta and GH3 cells. Using electron spin resonance (ESR) and the biologically active spin-labeled T3 (SL-T3), the transverse motion (flip-flop) of T3 in dipalmitoylphosphatidyl choline (DPPC) membranes was examined. The results indicate that SL-T3 does not flip-flop at any appreciate rate in the membranes. The data suggest that once partitioned into a cell membrane, T3 would remain in the outer half of the lipid bilayer. This result diminishes the possibility that T3 enters the cell by passive diffusion and further strengthens the conclusion that the entry of T3 into cells occurs by a receptor-mediated process.